U.S. patent number 11,179,445 [Application Number 16/326,210] was granted by the patent office on 2021-11-23 for pharmaceutical composition and biomaterial comprising fusion peptide in which bone tissue-selective peptide bound to parathyroid hormone (pth) or fragment thereof.
This patent grant is currently assigned to NANO INTELLIGENT BIOMEDICAL ENGINEERING CORPORATION CO. LTD., SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION. The grantee listed for this patent is Nano Intelligent Biomedical Engineering Corporation Co. Ltd., Seoul National University R&DB Foundation. Invention is credited to Chong-Pyoung Chung, Jue-Yeon Lee, Yoon Jeong Park.
United States Patent |
11,179,445 |
Park , et al. |
November 23, 2021 |
Pharmaceutical composition and biomaterial comprising fusion
peptide in which bone tissue-selective peptide bound to parathyroid
hormone (PTH) or fragment thereof
Abstract
The present invention relates to a pharmaceutical composition
for preventing or treating bone diseases comprising a fusion
peptide in which a bone tissue-selective peptide bound to
parathyroid hormone (PTH) or a fragment thereof. More particularly,
the present invention relates to a pharmaceutical composition and
biomaterial for preventing or treating bone diseases comprising a
fusion peptide in which a bone tissue-selective peptide represented
by an amino acid sequence of SEQ ID NO. 3 bound to parathyroid
hormone (PTH) or a fragment thereof represented by an amino acid
sequence of SEQ ID NO. 4 or 5. The fusion peptide can improve
effects of PTH by selectively binding to bone tissue and can reduce
administration frequency by increasing the half-life. The fusion
peptide can be used as a subcutaneous or intravenous injection-type
pharmaceutical composition for treating osteoporosis and fracture,
and can be used in combination with a medical device for tissue
recovery to increase formation of bone tissue. In addition, when
the fusion peptide is bound to the surface of dental and orthopedic
medical devices, transplantation stability of the medical device
can be improved through improved osseointegration between the
medical device and new bone.
Inventors: |
Park; Yoon Jeong (Seoul,
KR), Chung; Chong-Pyoung (Seoul, KR), Lee;
Jue-Yeon (Gyeonggi-do, KR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Nano Intelligent Biomedical Engineering Corporation Co. Ltd.
Seoul National University R&DB Foundation |
Chungcheongbuk-do
Seoul |
N/A
N/A |
KR
KR |
|
|
Assignee: |
NANO INTELLIGENT BIOMEDICAL
ENGINEERING CORPORATION CO. LTD. (Chungcheongbuk-Do,
KR)
SEOUL NATIONAL UNIVERSITY R&DB FOUNDATION (Seoul,
KR)
|
Family
ID: |
1000005952220 |
Appl.
No.: |
16/326,210 |
Filed: |
November 29, 2017 |
PCT
Filed: |
November 29, 2017 |
PCT No.: |
PCT/KR2017/013749 |
371(c)(1),(2),(4) Date: |
February 17, 2019 |
PCT
Pub. No.: |
WO2019/066140 |
PCT
Pub. Date: |
April 04, 2019 |
Prior Publication Data
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|
|
Document
Identifier |
Publication Date |
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US 20200113978 A1 |
Apr 16, 2020 |
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Foreign Application Priority Data
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Sep 29, 2017 [KR] |
|
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10-2017-0126876 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L
27/56 (20130101); A61K 35/32 (20130101); A61L
27/06 (20130101); A61K 38/1709 (20130101); A61K
38/39 (20130101); A61K 38/29 (20130101); A61L
27/10 (20130101); A61L 27/24 (20130101); A61L
27/3608 (20130101); A61L 27/3641 (20130101); A61L
2430/02 (20130101) |
Current International
Class: |
A61K
38/00 (20060101); A61K 38/29 (20060101); A61L
27/24 (20060101); A61K 38/17 (20060101); A61L
27/56 (20060101); A61L 27/36 (20060101); A61K
35/32 (20150101); A61L 27/10 (20060101); A61L
27/06 (20060101); A61K 38/39 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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105924646 |
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Sep 2016 |
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CN |
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107056794 |
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Aug 2017 |
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CN |
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07122118 |
|
May 1995 |
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JP |
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2010523671 |
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Jul 2010 |
|
JP |
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2013129705 |
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Jul 2013 |
|
JP |
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2015167906 |
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Sep 2015 |
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JP |
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2015167906 |
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Sep 2020 |
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JP |
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101183262 |
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Sep 2012 |
|
KR |
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1020130031870 |
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Mar 2013 |
|
KR |
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2010092135 |
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Aug 2010 |
|
WO |
|
2011090086 |
|
Jul 2011 |
|
WO |
|
Other References
Baht et al. Bone sialoprotein-collagen interaction promotes
hydroxyapatite nucleation. Matrix Biology 27 (2008) 600-608 (Year:
2008). cited by examiner .
Office of the Surgeon General (US). Tommy Thompson. Bone Health and
Osteoporosis: A Report of the Surgeon General (Year: 2004). cited
by examiner .
Rauch et al. Osteogenesis imperfecta. The Lancet vol. 363, Issue
9418, Apr. 24, 2004, pp. 1377-1385 (Year: 2004). cited by examiner
.
Rodan et al. Therapeutic Approaches to Bone Diseases. Science Sep.
1, 2000: vol. 289, Issue 5484, pp. 1508-1514 (Year: 2000). cited by
examiner .
Britannica--Bone diseases. Accessed Nov. 12, 2020 at
https://www.britannica.com/browse/Bone-Diseases/1 (Year: 2020).
cited by examiner .
Healthline. Accessed on Nov. 12, 2020 at
https://www.healthline.com/health/osteogenesis-imperfecta#types
(Year: 2020). cited by examiner .
cancer.gov. National cancer institute. Accessed on Nov. 12, 2020 at
https://www.cancer.gov/types/bone/bone-fact-sheet (Year: 2020).
cited by examiner .
Ponnapakkam, T., et al., "A Single Injection of the Anabolic Bone
Agent, Parathyroid Hormone-Collagen Binding Domain (PTH-CBD),
Results in Sustained Increases in Bone Mineral Density for up to 12
Months in Normal Female Mice", "Calcif. Tissue Int.", 2012, pp.
196-203, vol. 91. cited by applicant .
Genbank, "secreted phosphoprotein 1 (osteopontin, bone sialoprotein
1, early T-lymphocyte activation 1), partial [synthetic
construct]", Jul. 26, 2016, Version AAV38943.1. cited by applicant
.
Ponnapakkam, T., et al., "Monthly Administration of a Novel
PTH-Collagen Binding Domain Fusion Protein is Anabolic in Mice",
"Calcified Tissue International", 2011, pp. 511-520, vol. 88,
Publisher: Springer Science+Business Media, LLC. cited by applicant
.
Wu, X-C, et al., "Collagen-targeting parathyroid hormone-related
peptide promotess collagen binding and in vitro chondrogenesis in
bone marroa-derived MSCs", "International Journal of Molecular
Medicine", vol. 31, 2013, pp. 430-436. cited by applicant .
Young, M.F., "Bone matrix proteins: their function, regulation, and
relationship to osteoporosis", "Osteoporos Int.", 2003, pp.
S35-S42, vol. 14, No. Suppl 3, Publisher: International
Osteoporosis Foundation and national Osteoporosis Foundation. cited
by applicant.
|
Primary Examiner: Alstrum-Acevedo; James H
Assistant Examiner: Sabila; Mercy H
Attorney, Agent or Firm: Hultquist, PLLC Hultquist; Steven
J.
Claims
The invention claimed is:
1. A method for treating osteoporosis by selectively distributing a
fusion peptide to bone tissue, comprising administering (i) the
fusion peptide in which a bone tissue-selective peptide represented
by an amino acid sequence of SEQ ID NO: 3 bound to parathyroid
hormone (PTH) or a fragment thereof, as an active ingredient; or
(ii) a composition comprising said fusion peptide, wherein the
content of the fusion peptide in the composition is 10 to 100
.mu.g.
2. The method for treating osteoporosis according to claim 1,
wherein the fusion peptide induces formation of bone tissue.
3. The method for treating osteoporosis according to claim 1,
wherein the parathyroid hormone (PTH) is represented by an amino
acid sequence of SEQ ID NO. 4.
4. The method for treating osteoporosis according to claim 1,
wherein the fragment is represented by an amino acid sequence of
SEQ ID NO. 5.
5. The method for treating osteoporosis according to claim 1,
wherein the bone tissue-selective peptide is derived from bone
sialoprotein I.
6. The method for treating osteoporosis according to claim 1,
wherein the fusion peptide has a structure in which the N-terminus
of the bone tissue-selective peptide is bound to a C-terminus of
parathyroid hormone (PTH) or a fragment thereof.
7. The method for treating osteoporosis according to claim 1,
wherein the composition is formulated for intravenous,
intraperitoneal, intramuscular, intraarterial, oral, paradental,
intracardial, intramedullary, intrathecal, transdermal, intestinal,
subcutaneous, sublingual or topical administration.
8. The method for treating osteoporosis according to claim 1,
wherein the composition is formulated into any one selected from
the group consisting of injections, oral mucosal agents, capsules,
films, patches, percutaneous agents and gels.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. national phase under the provisions of
35 U.S.C. .sctn. 371 of International Patent Application No.
PCT/KR17/13749 filed Nov. 29, 2017, which in turn claims priority
of Korean Patent Application No. 10-2017-0126876 filed Sep. 29,
2017. The disclosures of International Patent Application No.
PCT/KR17/13749 and Korean Patent Application No. 10-2017-0126876
are hereby incorporated herein by reference in their respective
entireties, for all purposes.
TECHNICAL FIELD
The present invention relates to a pharmaceutical composition and
biomaterial for preventing or treating bone diseases comprising a
fusion peptide in which a bone tissue-selective peptide bound to
parathyroid hormone (PTH) or a fragment thereof.
BACKGROUND
Parathyroid hormone (PTH) is a peptide hormone composed of 84 amino
acids secreted from the parathyroid gland. PTH, which primarily
acts on the adrenal cortex, is a physiologically active substance
that binds to the adrenal cortex and increases the production of
cAMP, inositol triphosphate (IP3), and diacyl glycerol (DAG). PTH
increases calcium concentration in the blood by increasing
absorption of calcium in the bone and kidney. In addition, PTH,
which is intermittently administered, stimulates osteoblasts to
induce bone formation.
Therapeutic agents for osteoporosis such as estrogen, calcitonin
and bisphosphonate have a mechanism to inhibit bone resorption
(osteolysis), while PTH has a mechanism to promote bone formation
(osteogenesis). Drugs for inhibiting bone resorption are not
sufficient to increase the bone amounts of patients with advanced
osteoporosis already, but PTH has a mechanism to directly promote
osteogenesis and is thus beneficial to patients with type
osteoporosis with reduced bone remodeling or already advanced
osteoporosis. Currently, Forteo.RTM. commercially available from
Eli Lilly and Company, is known as a product approved as a
therapeutic agent for osteoporosis, which uses a peptide consisting
of 34 amino acids at the N-terminus among the 84 amino acids of
PTH. However, Forteo.RTM. is administered by subcutaneous injection
once a day due to its short half-life of 1 hour or less and thus
has low patient compliance. In addition, Forteo.RTM. may cause side
effects such as hypercalcemia and even a high incidence of
osteosarcoma, upon use for a long time of 2 years or longer. For
this reason, the use thereof for more than 2 years is
prohibited.
There have been attempts to increase the stability of PTH. For
example, PEG (U.S. Pat. No. 6,506,730) or albumin (WO 2010/092135)
is linked to PTH to induce long circulation in blood, or amino acid
is substituted to reduce the degradation by enzymes (KR
10-1183262).
In addition, there have been attempts to introduce physiologically
active factors to improve bone regeneration (osteoanagenesis) and
bone integration (osseointegration) of medical devices used in
dentistry and orthopedics. Medical devices used in dentistry and
orthopedics include bone grafts, barrier membranes, composite
materials containing collagen, metal implants, screws and the like.
However, since physiologically active factors are released from the
surface and decomposed, the effects thereof are insufficient.
Accordingly, as a result of intensively attempted research to solve
the aforementioned problems of the prior art, the present inventors
developed a pharmaceutical composition and biomaterial comprising a
fusion peptide in which a bone-tissue selective peptide bound to
PTH or a fragment thereof, and found that the pharmaceutical
composition comprising the fusion peptide is effective for the
treatment of conditions requiring osteoanagenesis such as
osteoporosis and fracture, and the fusion peptide is bound to the
surface of a dental and orthopedic medical device and is then
transplanted to increase the effect of osteoanagenesis, thereby
completing the present invention.
The information disclosed in the Background section is provided
only for better understanding of the background of the present
invention, and it is not intended to include information creating
the prior art already known to those skilled in the art.
DISCLOSURE
Technical Problem
Therefore, it is one object of the present invention to provide a
pharmaceutical composition for preventing or treating bone diseases
that comprises a fusion peptide with improved stability,
selectivity to bone tissue and bone regeneration (osteoanagenesis)
effect, as an active ingredient.
It is another object of the present invention to provide a method
for preventing or treating bone diseases comprising administering a
composition comprising a fusion peptide with improved stability,
selectivity to bone tissue and bone regeneration effect, as an
active ingredient.
It is another object of the present invention to provide the use of
a composition comprising a fusion peptide with improved stability,
selectivity to bone tissue and bone regeneration effect, as an
active ingredient, for preventing or treating bone diseases.
It is yet another object of the present invention to provide a
biomaterial in which a fusion peptide with improved stability,
selectivity to bone tissue and bone regeneration effect, bound
thereto.
Technical Solution
In order to achieve the foregoing objects, the present invention
provides a pharmaceutical composition for preventing or treating
bone diseases comprising a fusion peptide in which a bone
tissue-selective peptide bound to parathyroid hormone (PTH) or a
fragment thereof, as an active ingredient.
In addition, the present invention provides a method for preventing
or treating bone diseases comprising administering a composition
comprising a fusion peptide in which a bone tissue-selective
peptide bound to parathyroid hormone (PTH) or a fragment thereof,
as an active ingredient.
Further, the present invention provides the use of a composition
comprising a fusion peptide in which a bone tissue-selective
peptide bound to parathyroid hormone (PTH) or a fragment thereof,
as an active ingredient, for preventing or treating bone
diseases.
In addition, the present invention provides a biomaterial linked a
fusion peptide in which a bone-tissue selective peptide bound to
parathyroid hormone (PTH) or a fragment thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
FIG. 1 is a graph showing the concentration of PTH and a fusion
peptide in which a bone tissue-selective peptide bound to PTH in
blood, (A) when injected intravenously and (B) when injected
subcutaneously, wherein .box-solid. represents PTH and
.circle-solid. represents a fusion peptide in which a bone
tissue-selective peptide bound to PTH);
FIG. 2 is a microCT image of the femur of osteoporosis-induced mice
(A), and shows results of measurement of bone mineral density (BMD)
(B), after injection of PTH and a fusion peptide in which a bone
tissue-selective peptide bound to PTH into the osteoporosis-induced
mice, wherein .box-solid. represents no treatment, .circle-solid.
represents PTH and .tangle-solidup. represents a fusion peptide in
which a bone tissue-selective peptide bound to PTH);
FIG. 3 is a result of measurement of calcium concentration in blood
after injection of PTH and a fusion peptide in which a bone
tissue-selective peptide bound to PTH into osteoporosis-induced
mice, wherein .box-solid. represents no treatment, .circle-solid.
represents PTH and .tangle-solidup. represents a fusion peptide in
which a bone tissue-selective peptide bound to PTH);
FIG. 4 shows results of histological and histomorphometric
observation regarding new bone after transplanting a bone graft
comprising PTH and a fusion peptide in which a bone
tissue-selective peptide bound to PTH into a rabbit skull; and
FIG. 5 shows the distribution of a bone tissue near an implant of
fluorescence-labeled PTH and fluorescence-labeled fusion peptide in
which a bone tissue-selective peptide bound to PTH.
DETAILED DESCRIPTION OF THE INVENTION AND SPECIFIC EMBODIMENTS
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as those appreciated by those skilled
in the field to which the present invention pertains. In general,
nomenclature used herein is well-known in the art and is ordinarily
used.
In one embodiment of the present invention, it was found that bone
density was increased and bone generation effect was improved, as
compared to parathyroid hormone (PTH), by injecting a
pharmaceutical composition comprising a fusion peptide in which a
bone tissue-selective peptide bound to parathyroid hormone (PTH) or
a fragment thereof into osteoporosis-induced mice.
Accordingly, in one aspect, the present invention is directed to a
pharmaceutical composition for preventing or treating bone diseases
comprising a fusion peptide in which a bone tissue-selective
peptide bound to parathyroid hormone (PTH) or a fragment thereof,
as an active ingredient.
In another aspect, the present invention is directed to a method
for preventing or treating bone diseases comprising administering a
composition comprising a fusion peptide in which a bone
tissue-selective peptide bound to parathyroid hormone (PTH) or a
fragment thereof, as an active ingredient.
In another aspect, the present invention is directed to the use of
a composition comprising a fusion peptide in which a bone
tissue-selective peptide bound to parathyroid hormone (PTH) or a
fragment thereof, as an active ingredient, for preventing or
treating bone diseases.
According to the present invention, the fusion peptide induces
formation of bone tissue.
According to the present invention, the PTH is represented by an
amino acid sequence of SEQ ID NO. 4.
According to the present invention, the fragment is represented by
an amino acid sequence of SEQ ID NO. 5. SEQ ID NO. 4: SVSEIQLMH
NLGKHLNSME RVEWLRKKLQ DVHNFVALGA PLAPRDAGSQ RPRKKEDNVL VESHEKSLGE
ADKADVNVLT KAKSQ
SEQ ID NO. 5: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF
According to the present invention, the PTH or a fragment thereof
may be recombinant PTH expressed in E.coli or yeast, or a
PTH-related peptide (PTHrp) or may be produced by peptide synthesis
(Hefti et al., Clinical Science, 62, 389-396(1982); Liu et al., J.
Bone Miner. Res., 6: 10, 1071-1080(1991); Hock et al., J. Bone.
Min. Res., 7: 1. 65-71(1992)).
According to the present invention, the bone tissue-selective
peptide is represented by an amino acid sequence of SEQ ID NO.
3.
According to the present invention, the bone tissue-selective
peptide is derived from bone sialoprotein I, but is not limited
thereto.
According to the present invention, the peptide that imparts bone
tissue-selectivity to PTH is a peptide having binding force to
collagen, which is a main ingredient of bone. The bone tissue has a
structure in which mineral ingredients are deposited on collagen
fibers. Thus, the bone tissue-selective peptide facilities
migration of PTH into bone tissue.
In one embodiment of the present invention, the peptide imparting
bone tissue-selectivity used herein was separated and extracted
from the amino acid sequence of the active site in proteins
constituting the extracellular matrix, and was designed to maintain
the active structure through chemical modification after
extraction. Specifically, the peptide was required to comprise any
one of YGLRSKS (SEQ ID NO. 1), KKFRRPDIQYPDAT (SEQ ID NO. 2) and
YGLRSKSKKFRRPDIQYPDAT (SEQ ID NO. 3) amino acid sequences at the
positions of 149 to 169 of human bone sialoprotein I. To facilitate
chemical binding to PTH, cysteine was added in the form of a CGG-
or CGGGGG-spacer to the N-terminus of the amino acid sequence
selected from the amino acid sequences listed above and was
chemically synthesized to prepare the peptide.
According to the present invention, the fusion peptide may have a
structure in which the N-terminus of the bone tissue-selective
peptide is bound to the C-terminus of PTH or a fragment
thereof.
The bone tissue-selective peptide may be bound to the C-terminus of
PTH or a fragment thereof by solid phase peptide synthesis or with
the use of a chemical crosslinking agent, but the present invention
is not limited thereto.
According to the present invention, a chemical crosslinking agent
may be used to link the N-terminus of the bone tissue-selective
peptide to the C-terminus of PTH or a fragment thereof. In this
case, a functional group capable of binding to the cysteine at the
terminal of the peptide, for example, an SH group can be
introduced, or treatment can be performed to form amine (NH.sub.2),
thereby facilitating the subsequent cross-linking reaction using a
crosslinking agent.
The chemical crosslinking agent may be selected from the group
consisting of 1,4-bis-maleimidobutane (BMB),
1,11-bis-maleimidotetraethyleneglycol (BM[PEO].sub.4),
1-ethyl-3-[3-dimethyl aminopropyl] carbodiimide hydrochloride
(EDC),
succinimidyl-4-[N-maleimidomethylcyclohexane-1-carboxy-[6-amidocaproate]]
(SMCC) and sulfonates thereof (sulfo-SMCC), succinimidyl
6-[3-(2-pyridyldithio)-propionamido] hexanoate] (SPDP) and
sulfonates thereof (sulfo-SPDP),
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and sulfonates
thereof (sulfo-MBS), succinimidyl [4-(p-maleimidophenyl) butyrate]
(SMPB) and sulfonates thereof (sulfo-SMPB), but the present
invention is not limited thereto.
In order to remove the crosslinking agent after binding of PTH to
the bone tissue-selective peptide, the fusion peptide in which a
bone tissue-selective peptide bound to PTH is subjected to
purification such as ultrafiltration so that the fusion peptide in
which a bone tissue-selective peptide bound to PTH has a purity of
90% or more, more preferably, 98% or more.
In the present invention, the bone disease is selected from the
group consisting of osteoporosis, osteogenesis imperfecta,
hypercalcemia, osteomalacia, Paget's disease, bone loss and
osteonecrosis due to cancer, osteoarthritis, rheumatoid arthritis,
periodontal disease and fracture, but is not limited thereto.
In the present invention, the pharmaceutical composition for
preventing or treating bone diseases may be formulated for
intravenous, intraperitoneal, intramuscular, intraarterial, oral,
paradental, intracardial, intramedullary, intrathecal, transdermal,
intestinal, subcutaneous, sublingual or topical administration, but
is not limited thereto.
In the present invention, the pharmaceutical composition for
preventing or treating bone diseases is formulated into any one
selected from the group consisting of injections, oral mucosal
agents, capsules, films, patches, percutaneous agents and gels, but
is not limited thereto. The pharmaceutical composition may be
administered via topical, subcutaneous, intravenous, or parenteral
routes. In general, the pharmaceutical composition may contain a
therapeutically effective amount of the fusion peptide in which a
bone-tissue selective peptide bound to PTH or a fragment thereof,
as an active ingredient, according to the present invention.
In the present invention, the pharmaceutical composition may be
prepared by a well-known method using a pharmaceutically acceptable
inert inorganic or organic excipient. Examples of the excipient for
preparing injections include, but are not limited to, water,
alcohols, glycerol, polyols, vegetable oils and the like.
Alternatively, the injection may be used in combination with a
preservative, an analgesic agent, a solubilizer and a stabilizer.
The topical formulation may be prepared in the form of a gel or
film and the main ingredient of the gel is preferably collagen,
chitosan, hyaluronic acid, alginic acid, propylene glycol,
propylene glycol alginate, poloxamer, chondroitin sulfate or the
like.
In the present invention, the content of the fusion peptide in the
pharmaceutical composition may be 10 to 100 .mu.g. The
pharmaceutical composition may be formulated into a single
subcutaneous or intravenous injection containing a dose of 10 to
100 .mu.g of the fusion peptide in which a bone-tissue selective
peptide bound to PTH or a fragment thereof.
In one embodiment of the present invention, the effect of bone
regeneration can be identified using a bone implant linked the
fusion peptide in which a bone-tissue selective peptide bound to
PTH or a fragment thereof.
In another aspect, the present invention is directed to a
biomaterial linked the fusion peptide in which a bone-tissue
selective peptide bound to parathyroid hormone (PTH) or a fragment
thereof.
According to the present invention, the fusion peptide induces
formation of bone tissue.
According to the present invention, the PTH is represented by an
amino acid sequence of SEQ ID NO. 4.
According to the present invention, the fragment is represented by
an amino acid sequence of SEQ ID NO. 5.
According to the present invention, the bone tissue-selective
peptide is represented by an amino acid sequence of SEQ ID NO.
3.
According to the present invention, the bone-tissue selective
peptide has a structure in which the N-terminus of the bone
tissue-selective peptide is bound to the C-terminus of PTH or a
fragment thereof.
The bone tissue-selective peptide may be bound to the C-terminus of
PTH or a fragment thereof by a crosslinking agent. The crosslinking
agent may be selected from the group consisting of
1,4-bis-maleimidobutane (BMB),
1,11-bis-maleimidotetraethyleneglycol (BM[PEO].sub.4),
1-ethyl-3-[3-dimethyl aminopropyl] carbodiimide hydrochloride
(EDC),
succinimidyl-4-[N-maleimidomethylcyclohexane-l-carboxy-[6-amidocaproate]]-
(SMCC) and sulfonates thereof (sulfo-SMCC), succinimidyl
6-[3-(2-pyridyldithio)-propionamido] hexanoate](SPDP) and
sulfonates thereof (sulfo-SPDP),
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and sulfonates
thereof (sulfo-MBS), succinimidyl [4-(p-maleimidophenyl) butyrate]
(SMPB) and sulfonates thereof (sulfo-SMPB), but the present
invention is not limited thereto.
According to the present invention, the biomaterial may be any one
selected from the group consisting of bone grafts, barrier
membranes, implants and polymer scaffolds.
The biomaterial may include all kinds and types of bone grafts,
barrier membranes, implants and polymer scaffolds.
The bone graft comprises, as a main ingredient, an organism-derived
bone-mineral powder derived from autogenous bone, bovine bone and
porcine bone, and a porous block thereof, a synthetic
hydroxyapatite powder and a porous block thereof, a tricalcium
phosphate powder and a porous block thereof, or a mixture of
hydroxyapatite and tricalcium phosphate powders, and a porous block
thereof.
The barrier membrane is preferably produced from collagen,
chitosan, gelatin, polylactide, polylactide glycolide or
polycaprolactone, but is not limited thereto.
The implant may be produced from titanium alloy, titanium oxide or
zirconia, but the present invention is not limited thereto. The
implant may include dental and orthopedic implants. The orthopedic
implants include orthopedic fixation plates, orthopedic bone
screws, orthopedic bone nails, and the like.
In the present invention, the fusion peptide may be present in an
amount of 1 to 10 mg with respect to the unit weight (1 g) of the
biomaterial. More preferably, the fusion peptide may be present in
an amount of 2 to 8 mg with respect to the unit weight (1 g) of the
biomaterial.
EXAMPLE
Hereinafter, the present invention will be described in more detail
with reference to examples. However, it is obvious to those skilled
in the art that these examples are provided only for illustration
of the present invention and should not be construed as limiting
the scope of the present invention.
Preparation Example 1
Preparation of Fusion Peptide in Which Bone Tissue-Selective
Peptide Bound to PTH by Solid Phase Peptide Synthesis
The peptide was synthesized using F-moc solid phase chemical
synthesis by connecting a bone tissue-selective peptide (SEQ ID NO.
3) and a PTH fragment (SEQ ID NO. 5) in order from the N-terminus.
The synthesized peptide sequence was cleaved from a resin, washed,
lyophilized, and then separated and purified by liquid
chromatography. The molecular weight of the purified peptide was
identified by MALDI-TOF assay.
Comparative Example 1
Preparation of PTH Fragment
The peptide was synthesized using F-moc solid phase chemical
synthesis by connecting a PTH fragment (SEQ ID NO. 5). The
synthesized peptide sequence was cleaved from a resin, washed,
lyophilized, and then separated and purified by liquid
chromatography. The molecular weight of the purified peptide was
identified by MALDI-TOF assay.
Preparation Example 2
Preparation of Fusion Peptide in Which Bone Tissue-Selective
Peptide Bound to PTH by Crosslinking Reaction
1 mg of the PTH fragment (SEQ ID NO. 5) was dissolved in 1 ml of a
conjugation reaction buffer (100 mM sodium phosphate, 150 mM sodium
chloride, 0.02% sodium azide, 1 mM EDTA). 40 .mu.l of a Sulfo-SMCC
(Thermo Scientific, 4.8 mg/ml) solution was added portionwise to
PTH in small amounts and reacted in the absence of light at room
temperature for 1 or hours. The unreacted sulfo-SMCC was removed by
ultrafiltration through a membrane with a molecular weight cut-off
of 500 kDa. A solution (1 mg/ml) of the peptide of SEQ ID NO. 3 in
a conjugation buffer was added thereto and the resulting mixture
was reacted in the absence of light for 4 to 8 hours. The fusion
peptide comprising the bone tissue-selective peptide bound to PTH
was subjected to ultrafiltration through a membrane with a
molecular weight cut-off of 3,000 kDa to remove the unreacted
peptide of SEQ ID NO. 3. Using MALDI-TOF and SDS-PAGE, the
molecular weight of the fusion peptide in which a bone
tissue-selective peptide bound to PTH was identified. The
theoretical molecular weight should be at least 6,701.89 kDa, when
taking into consideration the fact that the molecular weight of the
PTH fragment is 4,117.8 kDa, the molecular weight of the bone
tissue-selective peptide is 2,365 kDa, and the molecular weight
increased by Sulfo-SMCC is 219.09 kDa.
Preparation Example 3
Preparation of Pharmaceutical Composition Comprising Fusion Peptide
in which Bone Tissue-Selective Peptide Bound to PTH
A pharmaceutical composition comprising the fusion peptide in which
the bone tissue-selective peptide bound to PTH of Preparation
Example 2 as an active ingredient was prepared (Table 1).
TABLE-US-00001 TABLE 1 Pharmaceutical composition of Preparation
Example 3 Ingredient Weight (mg) Fusion peptide in which bone
tissue-selective peptide bound 1 to PTH Sodium chloride, USP 8.18
Sodium succinate 1.62 WFI 987.5 Sodium hydroxide, NF and/or acetic
acid, NF Total 1 g, pH 6
Comparative Example 2
Preparation of Pharmaceutical Composition Comprising PTH
A pharmaceutical composition comprising the PTH of Comparative
Example 1 as an active ingredient was prepared (Table 2).
TABLE-US-00002 TABLE 2 Pharmaceutical composition of Comparative
Example 2 Ingredient Weight (mg) PTH 1 Sodium chloride, USP 8.18
Sodium succinate 1.62 WFI 987.5 Sodium hydroxide, NF and/or acetic
acid, NF Total 1 g, pH 6
Preparation Example 4
Preparation of Bone Graft Linked Fusion Peptide in Which Bone
Tissue-Selective Peptide Bound to PTH
1 g of a bovine bone-derived bone graft was allowed to stand in
3-aminopropyl ethoxysilane (APTES, 1%) dissolved in hexane and then
washed three times with hexane. As a result, an amine residue was
formed on the surface and BMB as a crosslinking agent was added
thereto. 1 g of the bone graft particles bound to the crosslinking
agent were reacted with 20 mg of the fusion peptide in which a bone
tissue-selective peptide bound to PTH of Preparation Example 2 for
12 hours, washed 3 times with methanol and then washed 10 times
with purified water, to obtain a bone graft which the fusion
peptide in which a bone tissue-selective peptide bound to PTH is
fixed.
Comparative Example 3
Preparation of Bone Graft Comprising PTH Linked Thereto
1 g of a bovine bone-derived bone graft was allowed to stand in
3-aminopropyl ethoxysilane (APTES, 1%) dissolved in hexane and then
washed three times with hexane. As a result, an amine residue was
formed on the surface and BMB as a crosslinking agent was added
thereto. 1 g of the bone graft particles bound to the crosslinking
agent was reacted with 20 mg of the PTH of Comparative Example 1
for 12 hours, washed 3 times with methanol and then washed 10 times
with purified water to obtain a bone graft to which the PTH is
fixed.
Preparation Example 5
Preparation of Gel-Type Composition of Fusion Peptide in Which Bone
Tissue-Selective Peptide Bound to PTH
20 mg of the fusion peptide in which bone tissue-selective peptide
bound to PTH of Preparation Example 2 was homogeneously mixed with
1 ml of a 2% collagen solution and a syringe was filled with the
resulting mixture.
Comparative Example 4
Preparation of Gel-Type Composition of PTH
20 mg of the PTH of Comparative Example 1 was homogeneously mixed
with 1 ml of a 1 to 3% collagen solution and a syringe was filled
with the resulting mixture.
Example 1
Test for Determining Half-Life of Fusion Peptide in Which Bone
Tissue-Selective Peptide Bound to PTH
A composition comprising the PTH of Comparative Example 2 and a
composition comprising the fusion peptide in which a bone
tissue-selective peptide bound to PTH of Preparation Example 3 were
subcutaneously administered at a concentration of 100 .mu.g/kg to
SD (Sprague-Dawley) male rats (body weight 300-350g) and blood was
collected at 0, 2, 5, 10, 20, 30, 60, 180, 360, 720, and 1,440
minutes. In addition, the compositions were injected at a
concentration of 100 .mu.g/kg into the jugular vein, blood was
collected at 0, 5, 10, 15, 30, 60, 90, 120, 180 and 360 minutes,
and the plasma was separated by centrifugation at 14,000 rpm for 10
minutes. The concentration of PTH was measured by enzyme-linked
immunosorbent assay (ELISA) (Immutopics, Inc., San Clemente,
Calif.).
FIG. 1 shows the concentration of the fusion peptide in which a
bone tissue-selective peptide bound to PTH in blood over time. When
subcutaneously injected, PTH was not detected after 360 minutes,
but the fusion peptide in which a bone tissue-selective peptide
bound to PTH was detected at up to 1,440 minutes. In the case of
intravenous injection, PTH was not measured after 180 minutes, but
the fusion peptide in which a bone tissue-selective peptide bound
to PTH was measured up to at 360 minutes. This means that the
half-life of the fusion peptide in which a bone tissue-selective
peptide bound to PTH is longer than that of PTH.
Example 2
Efficacy Test of Fusion Peptide in Qhich Bone Tissue-Selective
Peptide to PTH in Osteoporosis Animal
Six week-old ICR mice were anesthetized by intramuscular injection
using a mixture of 10 mg/kg of xylazine (Rompun.RTM., Bayer, Korea)
and 100 mg/kg of ketamine (Ketalar.RTM., Yuhan Co., Ltd., Korea)
and then the ovaries present below the bilateral kidneys were
entirely removed carefully. Suturing was performed by an ordinary
method and 3 mg/kg of gentamicin (Gentamycin.RTM., JW
Pharmaceutical Corporation, Korea) was intramuscularly
injected.
Three months after the ovariectomy, whether or not bone loss
occurred was checked. The pharmaceutical composition comprising PTH
of Comparative Example 2 was administered at 20 .mu.g/kg daily for
3 months, and the pharmaceutical composition comprising the fusion
peptide in which a bone tissue-selective peptide bound to PTH of
Preparation Example 3 was administered at 80 pg/kg weekly for 6
months. The change in bone density was evaluated, as compared with
a group not treated with osteoporosis.
FIG. 2 shows a microCT image and measurement results of bone
mineral density (BMD) of the femur after injection of the fusion
peptide comprising a bone tissue-selective peptide bound to PTH
into osteoporosis-induced mice. In the group with no treatment
after ovariectomy, bone density was reduced due to significant bone
loss. The group treated with the fusion peptide in which a bone
tissue-selective peptide bound to PTH showed an increase in bone
density, as compared to the group treated with PTH (FIG. 2(A)). As
a result of measurement of variation in BMD (bone mineral density),
meaning the total mineral content in the femoral head (FIG. 2(B)),
the variation in BMD in the group with no treatment after
ovariectomy was found to be decreased. The group treated with PTH
showed an increase in BMD variation at up to one month and a
decrease starting at two months. The fusion peptide in which a bone
tissue-selective peptide bound to PTH showed an increase in BMD at
up to 6 months. This means that the fusion peptide in which a bone
tissue-selective peptide bound to PTH is much more effective in
bone regeneration than PTH.
FIG. 3 shows the result of measurement of the concentration of
calcium in blood after injection of the fusion peptide in which a
bone tissue-selective peptide bound to PTH into
osteoporosis-induced mice. The concentration of calcium in blood
was determined using a QuantiChrom.TM. calcium assay kit (Bioassay
Systems, Hayward, Calif.). PTH increased the concentration of
calcium at one month, but the fusion peptide in which a bone
tissue-selective peptide bound to PTH did not increase the
concentration of calcium in blood. One of the effects of PTH on the
human body is to increase the concentration of calcium in blood by
affecting bones and kidneys. Therefore, long-term administration of
PTH causes side effects that induce hypercalcemia. However, it was
confirmed that the fusion peptide in which a bone tissue-selective
peptide bound to PTH did not act to increase the concentration of
calcium in blood, because it affected only bone tissue, not
affecting the kidneys.
Example 3
Test for Bone Regeneration of Fusion Peptide in Which Bone
Tissue-Selective Peptide Bound to PTH
A circular bone defect site having a diameter of 10 mm was formed
in the skull region of anesthetized rabbits (New Zealand white
rabbit, cuniculus) and 100 mg of the bone graft prepared in
Preparation Example 4 and Comparative Example 3 were transplanted
into the bone defect site. The periosteum and the skin were
double-sutured. Animals were sacrificed 3 weeks after
transplantation, the collected specimens were fixed in a formalin
solution, and the tissue was embedded to produce samples with a
thickness of 20 .mu.m. The prepared samples were stained with
hematoxylin-eosin to prepare undecalcified specimens. The prepared
specimens were observed with an optical microscope and imaged.
FIG. 4 shows results of histological and histomorphometric
observation regarding new bones after transplanting a bone graft
comprising the fusion peptide in which a bone tissue-selective
peptide bound PTH into a rabbit skull. The bone regeneration effect
of the fusion peptide in which a bone tissue-selective peptide
bound to PTH was increased more than PTH. Therefore, it is expected
that the bone graft having a surface linked the fusion peptide in
which a bone tissue-selective peptide bound to PTH is more
effective in bone regeneration than the bone graft to which PTH
binds.
Example 4
Bone Migration Test for PTH and Fusion Peptide in Which Bone
Tissue-Selective Peptide Bound to PTH
Cyanine 5.5 was bound to the fusion peptide in which a bone
tissue-selective peptide bound to PTH of Preparation Example 2, and
unreacted cyanine 5.5 was removed. The cyanine 5.5-labeled fusion
peptide in which a bone tissue-selective peptide bound to PTH was
prepared by the method in accordance with Preparation Example 5.
The PTH of Comparative Example 1 was bound to cyanine 5.5 and
unreacted cyanine 5.5 was removed. As a control group, cyanine
5.5-labeled PTH was prepared in accordance with the method of
Comparative Example 4.
An implant was transplanted 8 weeks after extraction of the teeth
of beagles, and 100 .mu.L of a collagen gel comprising the cyanine
5.5-labeled fusion peptide in which a bone tissue-selective peptide
bound to PTH was injected into the surgical site and sutured.
Animals were sacrificed 3 weeks after transplantation, the
collected specimens were fixed in a formalin solution and tissues
were embedded to prepare specimens with a thickness of 20 .mu.m.
The prepared specimens were stained with hematoxylin-eosin to
prepare undecalcified specimens. The prepared specimens were
observed with a confocal microscopy and imaged. The fluorescent
intensity per a predetermined unit area near the implant was
measured.
FIG. 5 shows the distribution of a bone tissue near an implant of
the fluorescence-labeled fusion peptide in which a bone
tissue-selective peptide bound to PTH. When the PTH was
transplanted, there was almost no fluorescence in the surrounding
bone tissue. However, in the case of the fusion peptide in which a
bone tissue-selective peptide bound to PTH, fluorescence
distributed in the surrounding bone tissue was observed. This
indicates that the fusion peptide in which a bone tissue-selective
peptide bound to PTH selectively binds to bone tissue, as compared
to PTH.
INDUSTRIAL APPLICABILITY
According to the present invention, it is possible to increase the
selectivity to bone tissue and to increase the bone regeneration
effect by introducing a peptide having selectivity to bone tissue
into PTH or a fragment thereof. In addition, the peptide can be
developed into pharmaceutical compositions for preventing or
treating bone diseases, which can improve patient compliance, by
increasing the half-life of PTH and consequently increasing the
interval of administration. Further, the present invention is
useful for further improving bone regeneration effects by applying
PTH bound to a bone tissue-selective peptide to biomaterials for
dentistry and orthopedics.
Although specific configurations of the present invention have been
described in detail, those skilled in the art will appreciate that
this description is provided as preferred embodiments for
illustrative purposes and should not be construed as limiting the
scope of the present invention. Therefore, the substantial scope of
the present invention is defined by the accompanying claims and
equivalents thereto.
SEQUENCE LISTINGS
1
517PRTArtificial Sequencebone sialoprotein I 1Tyr Gly Leu Arg Ser
Lys Ser1 5214PRTArtificial Sequencebone sialoprotein I 2Lys Lys Phe
Arg Arg Pro Asp Ile Gln Tyr Pro Asp Ala Thr1 5 10321PRTArtificial
Sequencebone sialoprotein I 3Tyr Gly Leu Arg Ser Lys Ser Lys Lys
Phe Arg Arg Pro Asp Ile Gln1 5 10 15Tyr Pro Asp Ala Thr
20484PRTArtificial SequencePTH 4Ser Val Ser Glu Ile Gln Leu Met His
Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu
Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly Ala
Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys
Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly Glu
Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys65 70 75 80Ala Lys Ser
Gln534PRTArtificial SequencePTH 5Ser Val Ser Glu Ile Gln Leu Met
His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30Asn Phe
* * * * *
References